A signaling pathway involving the extracellular protein Reelin and the intracellular adaptor protein Disabled-1 (Dab1) controls cell positioning during mammalian brain development. Here, we demonstrate that Reelin binds directly to lipoprotein receptors, preferably the very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). Binding requires calcium, and it is inhibited in the presence of apoE. Furthermore, the CR-50 monoclonal antibody, which inhibits Reelin function, blocks the association of Reelin with VLDLR. After binding to VLDLR on the cell surface, Reelin is internalized into vesicles. In dissociated neurons, apoE reduces the level of Reelin-induced tyrosine phosphorylation of Dab1. These data suggest that Reelin directs neuronal migration by binding to VLDLR and ApoER2.

The gene mutated in reeler (reelin) encodes a protein secreted by neurons in the developing brain that controls laminar positioning of migrating cells in the CNS by an unknown mechanism. To investigate Reelin function, we used the nestin promoter to express Reelin ectopically in the ventricular zone and other brain regions in transgenic mice. In the presence of the endogenous protein, ectopic Reelin did not alter cell migration in the neocortex or the cerebellum. However, in the reeler background, ectopic Reelin induced tyrosine phosphorylation of Dab-1 in the ventricular zone and rescued some, but not all, of the neuroanatomic and behavioral abnormalities characteristic of reeler. These results indicate that Reelin does not function simply as a positional signal. Rather, it appears to participate in multiple events critical for neuronal migration and cell positioning.

Disabled (Dab) 1 and 2 are mammalian homologues of Drosophila DAB. Dab1 is a key cytoplasmic mediator in Reelin signaling that controls cell positioning in the developing central nervous system, whereas Dab2 is an adapter protein that plays a role in endocytosis. DAB family proteins possess an amino-terminal DAB homology (DH) domain that is similar to the phosphotyrosine binding/phosphotyrosine interaction (PTB/ PI) domain. We have solved the structures of the DH domains of Dab2 (Dab2-DH) and Dab1 (Dab1-DH) in three different ligand forms, ligand-free Dab2-DH, the binary complex of Dab2-DH with the Asn-Pro-X-Tyr (NPXY) peptide of amyloid precursor protein (APP), and the ternary complex of Dab1-DH with the APP peptide and inositol 1,4,5-trisphosphate (Ins-1,4,5-P 3 , the head group of phosphatidylinositol-4,5-diphosphate (PtdIns-4,5-P 2 )). The similarity of these structures suggests that the rigid Dab DH domain maintains two independent pockets for binding of the APP/ lipoprotein receptors and phosphoinositides. Mutagenesis confirmed the structural determinants specific for the NPXY sequence and PtdIns-4,5-P 2 binding. NMR spectroscopy confirmed that the DH domain binds to Ins-1,4,5-P 3 independent of the NPXY peptides. These findings suggest that simultaneous interaction of the rigid DH domain with the NPXY sequence and PtdIns-4,5-P 2 plays a role in the attachment of Dab proteins to the APP/lipoprotein receptors and phosphoinositide-rich membranes.The Drosophila disabled gene product (DAB) 1 was identified as the result of a genetic screen designed to isolate modifier genes of the abl tyrosine kinase (1-3). Defects in abl and disabled prevent the formation of proper axonal connections in the central nervous system and cause the death of flies during embryonic development. DAB is tyrosine-phosphorylated by the sevenless receptor kinase and functions as an adaptor protein to recruit SH2-SH3 domain proteins to the signaling complex in Drosophila (4). An evolutionarily conserved family of disabled proteins was revealed by the discovery of a 96-kDa growth factor-responsive phosphoprotein possessing an aminoterminal region of ϳ150 amino acids that is highly similar to the amino terminus of DAB (5). DAB family members contain an amino-terminal DAB homology (DH) domain that acts as a protein-protein and protein-phospholipid interaction module and is fused to diverse carboxyl-terminal sequences.The DH domain has been identified in two mammalian proteins, Dab1 (also called mDab) and Dab2 (also known as p96 or Doc-2) (5, 6). Dab1 plays a key role in migration of neurons during brain development as a component of the Reelin signaling pathway (7-14). Reelin, a large glycoprotein secreted by pioneer cell populations early in development (10, 11), directs the positioning of neurons during brain development by binding to very low density lipoprotein receptors or apolipoprotein E type 2 receptors (apoER2) (9, 12). Engagement of these receptors triggers tyrosine phosphorylation of Dab1 (9, 13-15), creating a scaffold that rec...

The study of mice with spontaneous and targeted mutations has uncovered a signaling pathway that controls neuronal positioning during mammalian brain development. Mice with disruptions in reelin, dab1, or both vldlr and apoER2 are ataxic, and they exhibit severe lamination defects within several brain structures. Reelin is a secreted extracellular protein that binds to the very low density lipoprotein receptor and the apolipoprotein E receptor 2 on the surface of neurons.

Microglia, the resident mononuclear phagocytic cells, are critical for immune responses within the CNS. They recognize and are activated by various Pathogen Associated Molecular Patterns (PAMPs). β‐glucans are PAMPs present within fungal cell walls that are known to trigger protective responses in a number of immune cells. To better understand microglial responses to β‐glucans and the underlying signaling pathways, we sought to determine if Dectin‐1, a major β‐glucan receptor recently identified in leukocytes, mediates β‐glucan induced activation of microglia. Here, we report that Dectin‐1 is expressed on the surface of murine primary microglia, and engagement of the receptor with particulate β‐glucan resulted in an increase in tyrosine phosphorylation of Syk, a hallmark feature of the Dectin‐1 signaling pathway. Moreover, phagocytosis of β‐glucan particles and subsequent intracellular production of ROS were also mediated by Dectin‐1. Further, interaction of β‐glucan with Dectin‐1 resulted in activation of Vav and PI3K/Akt pathways, suggesting that β‐glucan induce multiple signaling pathways in microglia. However, unlike in leukocytes, β‐glucan mediated microglial activation did not result in production of cytokines. Thus, the interaction of microglial Dectin‐1 with glucan elicits a unique response, suggesting that the Dectin‐1 pathway may play an important role in antifungal immunity in the CNS.